Hemostasis (the process of initiating blood coagulation in order to stop bleeding) may be abnormal and possibly life-threatening under a number of circumstances, yet there is a need for improved methods of inducing hemostasis under special conditions. Hemostasis is initiated by platelets, which are blood cells that cause occlusion of holes in blood vessels. In the course of doing so, platelets become “activated”. One aspect of their activation is the translocation of phosphatidylserine (PS) from the inner half of their plasma membrane to the outer half. Once this occurs, certain blood coagulation proteins bind to the surface of the platelet and interact in the process known as “blood coagulation”. The end result of that process is the formation of the enzyme thrombin (also called Factor IIa). Thrombin has several important roles. First, it converts the blood protein fibrinogen into fibrin. Each fibrin binds to several other fibrin molecules, forming a fibrin clot, which supports the platelets that are attempting to stop bleeding. Second, thrombin also activates platelets, increasing the number involved in the hemostatic process. And third, thrombin affects other plasma proteins, such as Factor XI, in a manner that accelerates the biochemistry of blood coagulation. The first proteins that interact on the surface of platelets (or other cells with exposed PS) are Tissue Factor (TF), the activated form of Factor VII (called Factor VIIa), and Factor X.
Thrombin generation is initiated by the interaction of the plasma serine protease, Factor VIIa, with its protein cofactor, TF. TF is a membrane-bound protein not expressed on the surface of cells in contact with the bloodstream until they become activated. Upon its expression, TF binds either Factor VII (promoting its activation to Factor VIIa), or Factor VIIa, increasing its catalytic efficiency in converting Factor X to Factor Xa. Expression of the extracellular domain of TF (amino acids 1-219, or a 3-219 residue portion of the entire protein) in E. coli generates a polypeptide of about 26 kDa that retains the ability to bind to Factor VIIa and to allosterically activate it. This truncated TF (called soluble TF or sTF) does not bind to cellular membranes and is therefore generally much less efficient than native TF in promoting Factor VII autoactivation or activation of Factor X by Factor VIIa. Engineering of the cDNA encoding sTF so that it was expressed on the surface of mammalian cells as a glycosylphosphatidylinositol-anchored protein resulted in a protein with the same specific procoagulant activity as native TF, underscoring the importance of membrane attachment for this protein.
It has long been recognized that congenital Factor VIII deficiency is characterized by abnormal thrombin generation when blood coagulation is triggered by low concentrations of TF. More recently recognized is the fact that disorders of platelet function are also associated with decreased thrombin generation. The vitamin K-dependent blood coagulation proteins, which are required for thrombin generation, assemble on the surface of activated platelets, endothelial cells, and/or monocytes by binding to anionic phospholipids, especially PS. Thrombin, a potent platelet agonist, amplifies the activation of platelets initiated by contact with sub-endothelial collagen exposure. Delayed thrombin generation may therefore underlie or amplify the bleeding tendency accompanying disorders of plasma coagulation factors, or blood platelets.
Others have reported that the intravenous injection of thromboplastin (membrane-bound TF) into animals results in generalized activation of the coagulation, as does injection of both sTF and Factor VIIa, resulting in beneficial effects upon bleeding in experimental animals, suggesting that sTF might serve as the basis of a therapy designed to reduce bleeding.
A therapeutic protein effective in enhancing hemostasis and coagulation in a subject in need thereof would be desirable.